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Identification and Characterization of Novel CRISPR-Cas Immune Effectors
No full textBacteria are under constant threat by the viruses that infect them called bacteriophages or phages for short. It is predicted that phages outnumber bacteria by an order of magnitude, producing a tremendous pressure for resistance mechanisms against this threat. One of the most abundant immune systems bacteria have evolved to combat this are CRISPR-Cas systems, which have been studied extensively due to their fascinating biology and usefulness in biotechnology and therapeutics. My thesis focuses on the discovery and characterization of three new CRISPR-Cas effector proteins that provide immunity in bacteria in response to phage challenge with highly diverse activities. CRISPR-Cas systems are the only known adaptive immune system found in bacteria. Immunity by these systems is achieved in two main steps: (1) immunization, which is the process of acquisition of small sequences of nucleic acids from the invader into CRISPR arrays as \u27spacers\u27 on the host chromosome as immunological memories of pathogens and (2) interference, which is the process of the transcription and maturation of CRISPR arrays into crRNAs containing spacer sequences that are subsequently loaded onto a CRISPR effector protein or protein complex that survey nucleic acids for sequences complementary to transcribed spacer sequences (protospacers). Upon base pairing between crRNAs and protospacers, the Cas machinery mediates destruction of invader nucleic acids or initiates a different immune response. My thesis work has focused on a specific type of CRISPR systems called type III CRISPR systems. These CRISPR systems are unique in that they recognize invader RNA transcripts rather than DNA (which is more common) and upon recognition two activities are unleashed for immunity. The first is destruction of ssDNA by the Cas10-Csm complex, specifically via the catalytic activity of the HD domain within the Cas10 protein. Second, and exclusive to type III CRISPR, is the synthesis of cyclic oligoadenylate molecules (cOAs) of various sizes from ATP substrates by the catalytic activity of the palm domain. cOAs go on to activate additional accessory immune effectors encoded within type III CRISPR loci that provide immunity primarily through the activation of some toxic activity via their effector domains. The most common protein domain among these effectors is the CARF domain. This protein domain is responsible for receiving the cOA signals that Cas10- Csm makes upon sensing an infection in the cell. In my doctoral studies I have studied three proteins containing CARF domains fused to various other domains that are activated upon binding cOAs. The first protein I characterized, called cyclic oligoadenylate-activated membrane protein 1 (Cam1) contains a CARF domain fused to single a transmembrane helix. This protein was first identified as active in our model CRISPR system in a screen by a former member of the Marraffini laboratory, Jakob Rostøl. I determined that it mediates membrane depolarization by forming a tetrameric pore in the membrane of cells that likely opens when it binds a cOA. After this, I identified two additional proteins that were active in our CRISPR system, a CARF-adenosine deaminase protein fusion called CRISPR-associated adenosine deaminase 1 (Cad1), which deaminates ATP to ITP upon cOA binding and a CARFTIR protein fusion called CRISPR-associated TIR protein 1 (Cat1), which hydorlyzes NAD+ molecules upon cOA binding. I was able to identify these proteins by leveraging new bioinformatic tools developed during my PhD and characterize their molecular mechanisms. At the start of my doctoral studies, only type III CRISPR accessory proteins containing nuclease domains were characterized. My work has extensively expanded the mechanistic diversity in effector activities within the CRISPR-Cas immune response in bacteria. Additionally, these proteins offer potential uses in diagnostic tools, as their enzymatic responses can be stimulated by the presence of a RNA molecule of interest coupled with a crRNA-loaded Cas10-Csm complex and read out with relatively simple assays
Mechanisms of Telomere End Protection by TRF2
No full textShelterin is a six-subunit protein complex that protects the ends of mammalian chromosomes, called telomeres. The six subunits of shelterin, TRF1, TRF2, TIN2, TPP1, POT1, and Rap1, each have distinct roles in telomere homeostasis. These include telomere length regulation and preventing telomeres from activating various DNA damage response (DDR) signaling and double-strand break repair pathways. The need to suppress these DDR pathways at telomeres is known as the end protection problem. The TRF2 subunit of shelterin is responsible for suppressing the ATM DNA damage response kinase, which is activated by exposed DNA ends. TRF2 is also necessary and sufficient for forming t-loops, lariat-like structures that sequester the ends of telomeric DNA. In the absence of TRF2, t-loops are lost, ATM is activated, and telomeres fuse together via cNHEJ. While t-loops have been presented as an elegant solution to the end protection problem, it is yet unproven whether it is the t-loop itself that suppresses the DNA damage response, or whether other activities of TRF2 are equally or more important for end protection. There is evidence for the importance of both. The loss of t-loops coincides with ATM activation, suggesting an essential role for t-loops in ATM suppression. However, some work suggests that TRF2 can suppress ATM and the double-strand break repair pathway cNHEJ via t-loop-independent mechanisms. The ultimate proof that t-loops are necessary and sufficient for telomere end protection requires engineering of a TRF2-independent t-loop. This was previously unachievable because thus far, the mechanism of t-loop formation by TRF2 has been unknown. Since the discovery of t-loops 2.5 decades ago, the mechanism of t-loop formation has been a major focus in the field of telomere biology. Here, we propose a mechanism for t-loop formation by TRF2 and generate TRF2-independent t-loops in order to answer these longstanding questions. We first show that TRF2 possesses two dimerization domains and binds telomeric DNA as a tetramer. TRF2 has one dimerization domain in its TRFH that was discovered previously, and we identify a second one formed by regions in its Hinge and Myb domains. Tetramerization is necessary for t-loop formation by TRF2, as a TRF2 mutant lacking part of its second dimerization domain properly localizes to telomeres but is deficient in suppressing ATM and cNHEJ. The ability of this mutant to suppress ATM and cNHEJ was improved by artificial tetramerization. We demonstrate the sufficiency of tetramerization for t-loop formation by showing that TRF1, a dimeric protein that normally does not form t-loops and plays no role in end protection, forms t-loops when induced to tetramerize. Using this system to create t-loops in the absence of TRF2, we show that t-loops themselves are sufficient to block both ATM signaling and cNHEJ. Alt-EJ, however, was not suppressed in this system, indicating that TRF2 suppresses alt-EJ via a mechanism other than tetramerization and t-loop formation. We also investigate TRF2\u27s other means of protecting chromosome ends and show that the iDDR, a region within TRF2\u27s Hinge domain, suppresses both ATM signaling and the 53BP1 recruitment downstream of ATM signaling. This likely occurs via two independent mechanisms, one in which the iDDR inhibits ATM through an interaction with MRN, and another in which it recruits additional factors that disrupt the accumulation of 53BP1 downstream of ATM signaling. Additionally, Rap1, a shelterin component brought to the telomere by TRF2, suppresses cNHEJ. This may occur through binding of Rap1\u27s BRCT domain to Ku, blocking the interaction with Ligase 4 that is required for the completion of cNHEJ. TRF2 employs several mechanisms to suppress ATM and cNHEJ, including t-loop formation, thereby protecting chromosome ends from aberrant DNA damage signaling and repair. We propose that for most of the cell cycle, t-loops serve as the main mechanism of end protection by TRF2. Directly after leading-end DNA replication, the ends of the telomere must be resected to form the 3\u27 overhang before t-loops can form. During this resection process and delay in t-loop formation, we propose that the iDDR and Rap1 serve to suppress ATM and cNHEJ. Together, our results describe the intricate, elegant, and often redundant mechanisms of end protection by the essential and multifaceted shelterin component, TRF2
Mapping the Brain of the Clonal Raider Ant: Integrating Behavior, Neuroanatomy and Connectomics
No full textAnts have been studied extensively due to their advanced social structure and communication strategies. Olfaction plays a crucial role in their social behaviors, with ants using a variety of pheromones produced by different exocrine glands to communicate. For example, ants release alarm pheromones in response to danger to alert their nestmates and to trigger behavioral alarm responses. Despite extensive behavioral studies, the neurobiological mechanisms underlying these behaviors are not well understood. The clonal raider ant, Ooceraea biroi, is an ideal species for investigating the neural circuits involved in social behavior, as it is both experimentally tractable and genetically accessible compared to other ant species. In this thesis, I describe my efforts to bridge the gap between behavior and brain function in hopes of uncovering the neural underpinnings of social behavior in O. biroi. To begin, I focused on studying alarm behavior, as identification of a pheromone that triggers a robust, consistent, and conserved behavior, like the alarm pheromone, provides an avenue to dissect the behavioral and neuronal mechanisms underpinning chemical communication. I began by characterizing the alarm response of the clonal raider ant Ooceraea biroi. During an alarm response, ants quickly become unsettled, leave their nest pile, and are sometimes initially attracted to the source of alarm, but ultimately move away from it. We find that the alarm pheromone is released from the head of the ant and identify the putative alarm pheromone as a blend of two compounds found in the head, 4-methyl-3-heptanone and 4-methyl-3-heptanol. While 4-methyl-3-heptanone and 4-methyl-3-heptanol are known alarm pheromones in other more distantly related ant species, this is the first report of the chemical identity of a pheromone in O. biroi, and the first alarm pheromone identified in the genus Ooceraea. The synthetic alarm pheromone components have been used identify a core set of glomeruli within the antennal lobe of the ant that is active in response to panic alarm-inducing compounds, including 4-methyl-3-heptanone and 4-methyl-3-heptanol. We have also identified age-related changes in alarm behavior and processing of alarm pheromones in the antennal lobe, thereby linking neural function with behavior in the clonal raider ant. To advance neuroscience in the clonal raider ant, a deeper understanding of the brain\u27s gross anatomy is essential. To this end, we performed a comprehensive neuroanatomical analysis of the Ooceraea biroi brain to date, using immunohistochemistry, light microscopy, and advanced image processing techniques. We dissected, immunostained, and imaged the brains of forty age-matched, genetically identical individuals with confocal microscopy. Then, using 3D groupwise registration, we generated the first reference brain for the clonal raider ant. We use this new reference to conduct a 3D structural analysis of key regions putatively involved in regulating sociality and describe the neuroanatomy of major neurotransmitter systems in the clonal raider ant brain. Unexpectedly, we discovered extensive phenotypicstructural plasticity across our collection of brain samples. Half of the ants displayed a left-tilted brain anatomy, while the remaining half possessed a right-tilted \u27mirror image\u27 phenotype. In addition, 3D reconstructions revealed substantial variability in total neuropil volume across our dataset, with the most voluminous brains roughly three-fold larger than the smallest. These findings indicate clonal raider ants exhibit varied nervous system phenotypes despite apparent genotypic and experiential homogeneity. This work provides a powerful resource for the clonal raider ant neuroscience community, while simultaneously introducing novel features of the species\u27 neurobiology which may have important significance for social behaviors and colony function. Finally, to further investigate the structural connectivity of the O. biroi brain, we imaged a whole brain at synaptic-level resolution using transmission electron microscopy (TEM). This will allow us to reconstruct neurons, identify synapses, and ultimately create a complete wiring diagram, or connectome, of the clonal raider ant brain. To accelerate the mapping of neural circuits in the EM dataset, we used convolutional neural networks to segment neurons and predict synapses. To facilitate the matching of cells between EM and light microscopy, we have registered the EM volume to the clonal raider ant reference brain atlas. We are now in the process of manual proofreading of these automatic neuron segmentations to reconstruct neural circuits. Given the importance of olfaction in the pheromone-mediated communication of the ant and massive expansion of odorant receptor genes and olfactory glomeruli in the antennal lobe compared to other insects, our initial reconstruction efforts focus on understanding the wiring logic of the ant olfactory system. In summary, this thesis represents early steps toward understanding the neural circuits underlying social behavior in the clonal raider ant. The creation of a reference brain, along with ongoing efforts to map its connectome, contributes to the expanding toolkit for investigating the neural mechanisms that govern the complex social behaviors of ants
Serotonin-Mediated Sickness Behavior
No full textSerotonin is an ancient and powerful neuromodulator that regulates diverse processes from fundamental physiology to complex emotions. Due to the many functions of serotonin distributed throughout the body and brain, governing principles of serotonin signaling have remained elusive. There is particular interest in how serotonin modulates internal states given that serotonin dysregulation is a feature of many psychiatric diseases. In this thesis, I use the simplified serotonergic system of C. elegans to explore how serotonin governs the internal state of sickness. In addition to activating the immune system and remodeling physiology, pathogenic infection results in adaptive behavioral changes that promote survival. These \u27sickness behaviors\u27 are observed throughout the animal kingdom. I induced sickness in C. elegans by exposing them to a lawn of pathogenic bacteria, and then studied how avoidance behaviors developed over time. Animals slowly developed an aversion to the pathogen, and avoidance of a bacterial lawn over multiple hours. This behavior is delayed and reduced in mutants deficient in serotonin synthesis. I found that serotonin synthesis in two distinct neuronal cell types promotes pathogen avoidance at different stages of development. In L4-stage larvae, serotonin synthesized by NSM neurons in the pharynx increases avoidance of pathogenic bacteria. In adult animals, HSN neurons produce the serotonin required for acquired pathogen avoidance. Using a machine learning pipeline to track animals over a 20-hour assay, I characterized animals\u27 dynamic interactions with the edge of the pathogenic bacterial lawn. Serotonin synthesized by HSN neurons promotes exit and repulsion from the pathogenic bacteria and suppresses re-entry into the pathogen lawn. HSN neurons regulate egg-laying behavior, and when exposed to pathogen, animals are more likely to lay eggs in the absence of food than uninfected animals. These results suggest that in the pathogen context, HSN neurons are more active in the absence of bacteria. My results demonstrate that sickness-induced changes in serotonin signaling from neurons promote adaptive pathogen avoidance behavior
Molecular and Metabolic Regulation of Gastrointestinal Cancer Metastasis
No full textMost of the cancer related mortality caused by metastatic disease rather than primary tumor growth. More than 80% of gastrointestinal cancers initially metastasize to the liver first. While the prognosis of early-stage cancers is improving, a significant proportion of patients have metastatic disease at the time of diagnosis leading to poor survival outcomes. I focused on colorectal cancer and pancreatic cancer, the second and third leading causes of cancer related death in the United States, due to the lack of molecularly targeted therapies available for these cancers. Moreover, understanding the pathophysiological basis of gastrointestinal cancer liver metastasis is of great interest to the medical and scientific community. The first part of this thesis describes the establishment of multiple in vivo selected highly metastatic colorectal and pancreatic cancer models from poorly metastatic patient derive tumors and established cancer cell lines. Transcriptomic profiling of highly metastatic cancer cells identified phosphoenolpyruvate carboxykinase 1 (PCK1) in colorectal cancer and neuronal pentraxin 1 (NPTX1) in pancreatic cancer as potential metastasis promoters as they were highly differentially expressed in multiple highly metastatic models. The second part of this thesis first validates PCK1 and NPTX1 as metastasis promoters via in vivo functional assays followed by the mechanistic studies revealing PCK1\u27s and NPTX1\u27s role in promoting cancer cell growth under hypoxia, a hallmark of liver microenvironment. Metabolite profiling coupled with stable isotope tracing of highly metastatic colorectal cancer cells revealed that PCK1 reversed the TCA cycle under hypoxia to repurpose aspartate, a TCA cycle product, as a precursor of pyrimidines to promote hypoxic cell growth. On the other hand, in pancreatic cancer, NPTX1 was found to interact with adhesion molecule immunoglobulin like domain 2 (AMIGO2), newly identified cancer cell surface NPTX1 receptor in an autocrine manner that leads to nuclear retention of hypoxia inducible factor 1a (HIF1a) via phosphorylation of serine 641 and serine 643 residues. The final part of this study demonstrates therapeutic targeting of PCK1 and NPTX1-AMIGO2 pathways inhibits cancer metastasis and progression in multiple in vivo models. In collaboration with Tri-Institutional Therapeutics Discovery Institute, we developed an anti-NPTX1 therapeutic and diagnostic antibody that outperforms gemcitabine, a standard chemotherapeutic agent
Modulation of the CRISPR-Cas9 Immune Response
No full textBacteria face an onslaught of ever-changing viral (phage) invaders. Bacteria and phages are locked into an evolutionary arms race where both must continually adapt to survive. In response, bacteria have developed numerous immune systems to protect themselves, including CRISPR-Cas. CRISPR-Cas systems provide adaptive immunity through the acquisition of short DNA sequences from invading phages, known as spacers. Spacers are inserted into the CRISPR locus and serve as templates for the transcription of guides used by RNA-guided nucleases to recognize complementary sequences of the invaders and start the CRISPR immune response. CRISPR-Cas systems need to acquire enough spacers to mount an effective response against evolving threats, but not so many spacers as to risk autoimmunity through the acquisition of self-targeting spacers. In type II-A CRISPR systems, Cas9 uses the guide RNA to cleave target DNA sequences in the genome of infecting phages, and the tracrRNA to bind the promoter of cas genes and repress their transcription. We previously isolated a Cas9 mutant carrying the I473F substitution that enhanced spacer acquisition by 2-3 orders of magnitude, leading to a fitness cost due to higher levels of autoimmunity. Here we investigated the molecular basis of these findings. We found that the I473F mutation decreases the association of Cas9 to tracrRNA, limiting its repressor function, leading to high levels of expression of cas genes, which in turn strengthen the type II-A CRISPR-Cas immune response. I473 lines a conserved hydrophobic pocket that makes base-specific contacts with the nexus of tracrRNA, and substitutions within this pocket affect association of Cas9 to tracrRNA to modulate the repression of cas genes in related type II-A CRISPR-Cas systems. Our findings highlight the importance of the interaction between Cas9 and its tracrRNA cofactor in tuning the type II-A CRISPR-Cas immune response to balanced levels that enable phage defense but avoid autoimmunity
Investigating the Double-Edged Sword of Rifampicin Resistance in Mycobacterium Tuberculosis
No full textTuberculosis (TB) is the world\u27s deadliest infectious disease, though many in the rich world consider it an illness of Victorian nobility and poets. This is because, despite killing more than one million people annually, TB is most prevalent in low- and middle-income countries. TB is caused by the bacterium Mycobacterium tuberculosis (Mtb), and treatment for drug-sensitive TB consists of four antibiotics taken in combination for between four and six months. Treatment is long, costly, and is often inaccessible to those in TB endemic regions of the world. This pandemic is worsened by the emergence of drug resistant Mtb, particularly Mtb that is resistant to the first line antibiotic rifampicin (Rif). Rif resistant (RifR) Mtb caused up to 450,000 cases of TB and 264,000 deaths worldwide in 2021. Treatment for drug resistant TB is even more arduous, consisting of expensive second- and third-line antituberculars with side effects such as deafness, hepatotoxicity, and peripheral neuropathy, taken for six to nine months. RifR is caused by mutations in rpoB, the gene encoding the β subunit of RNA polymerase (RNAP), the target of Rif. These mutations block Rif binding but also alter the shape of the RNA exit channel, potentially disrupting transcription elongation. As is true for many other bacteria, drug resistance in Mtb is often associated with a fitness cost in the absence of antibiotics, and Rif resistance is no exception. RifR Mtb is frequently less fit than RifS Mtb and displays altered transcription dynamics, cell wall and lipid composition, and collateral sensitivity to some antibiotics. RifR Mtb is known to evolve second site compensatory mutations in the α and β\u27 subunits of RNAP that rescue the fitness of the strain to near Rif sensitive (RifS) levels. These compensatory mutations also ameliorate some of these biochemical and physiological defects of the resistant strain. A deeper understanding of the trade-offs encountered by RifR in Mtb is needed to develop effective treatment for resistant infections and potentially limit the evolution of drug resistance in a drug sensitive population. Here, we took a functional genomics approach to identify mechanisms underlying double-edged sword of RifR in βS450L Mtb, the most clinically common RifR conferring mutation, which accounts for over 70% of all RifR Mtb in the clinic. Employing genome-wide CRISPR interference (CRISPRi) screens, we identified genes with differential sensitivity to inhibition in RifR Mycobacterium smegmatis (Msmeg; a model organism of Mtb) and Mtb, compared to a RifS strain. We identified genes involved in translation-related processes as bolstering the fitness of RifR mycobacteria. This includes the essential transcription factor nusG, which has dual roles in promoting transcription elongation and transcription pausing/termination. Given the opposing roles of NusG, with the former potentially boosting the fitness of the slow βS450L RNAP, and the latter exacerbating the hyper-termination defect of βS450L RNAP, we investigated how nusG may be evolving in the clinic. Utilizing a genome-wide association study, we discovered novel mutations in nusG which buffer the fitness of βS450L Mtb by minimizing the pro-pausing activity of NusG. This ameliorates the slow transcription elongation and hyper-pausing/termination of βS450L Mtb. These results define hyper-termination as a source of the fitness defect of βS450L Mtb, identify a new series of compensatory mutations, and may inform new therapies to limit the evolution of drug-resistance. We also conducted differential vulnerability screening in two fast, hypo-terminating RifR mutants, βH445Y and βD435V Mtb. We found that many of the same genes and pathway that are more sensitive to inhibition in βS450L Mtb were less sensitive to inhibition in these fast RNAP mutants. For example, thiS, the gene encoding the thiamine diphosphate (TPP) synthase, is a top collateral vulnerability in βS450L Mtb, and a top collateral invulnerability in these fast, hypo-terminating RifR mutants. We determined that the enhanced vulnerability of thiS in βS450L Mtb is due to the depletion of branch chain amino acids (BCAA) upon TPP limitation. This led us to investigate ilvB1, the most differentially vulnerable TPP dependent enzyme in our dataset. ilvB1 encodes the large subunit of the acetohydroxyacid synthase enzyme, which catalyzes the first step of BCAA biosynthesis. Mtb ilvB1 gene expression is likely regulated by transcription attenuation, as it is in other bacteria. We utilized a series of reporter strains to determine that the inability of the slow, pause-prone βS450L RNAP to read through the regulatory terminator upstream of ilvB1 in the absence of BCAA plays a role in mediating the enhanced vulnerability of ilvB1. These data also highlight hyper-termination as a source of the fitness defect of βS450L Mtb and suggest that other genes controlled by attenuation mechanisms may be more vulnerable to inhibition in βS450L Mtb. The data presented in the text not only identify a new class of compensatory mutations in βS450L Mtb and define hyper-termination as a mediator of the fitness defect of βS450L but provide insights into the nature of other compensatory mutations and suggest rational ways to exacerbate the fitness cost of RifR Mtb for treatment and the prevention of the evolution of RifR. Known compensatory mutations in rpoA and rpoC, which encode the α and β\u27 subunits of RNAP, may rescue the fitness of βS450L Mtb by similar mechanisms as nusG and β protrusion clinical variants- that is by limiting the swiveling of the βS450L RNAP and minimizing hyper-termination. By targeting a collateral vulnerability, such as the pro-elongation activity of NusG or BCAA biosynthesis, one may be able to treat βS450L Mtb infections. Additionally, targeting a collateral vulnerability in tandem with Rif in a RifS population may be able to limit the evolution of RifR in the first place. Finally, these data suggest compounds that enhance transcription pausing and termination may also be able to treat RifR Mtb and shift the evolutionary path of resistance towards less fit RifR mutants that may be more easily cleared by the immune system
Exploring the Multifaceted Life of Beige Adipocytes
No full textObesity has become a major global health concern, with the number of individuals affected rising dramatically over the past several decades. Obesity is closely linked to a range of chronic diseases and contributes significantly to morbidity and mortality worldwide. Adipose tissue lies at the center of this issue. However, it is increasingly recognized that it is the quality of fat, rather than its quantity alone, that has a greater impact on metabolic health. While excess accumulation of lipid-storing white adipocytes is usually associated with metabolic complications, brown and beige adipocytes, specialized cell types that disspate energy through heat production, are associated with beneficial outcomes, including increased energy expenditure, improved glucose and lipid metabolism, and reduced odds of type 2 diabetes, hypertension, hyperlipidemia, and cardiovascular disease. These metabolic advantages make thermogenic adipocytes promising therapeutic targets for obesity and associated diseases. A deeper understanding of these cells could provide valuable insights for the development of more effective treatments for metabolic disease. In this study, we explored the multifaceted life of beige adipocytes. We charted their life trajectory from their emergence in early life to their persistence and function in adulthood. We found that beige adipocytes first emerge in life in a temperature-independent manner during the postnatal period. They later lose thermogenic activity as mice mature, but can be reactivated upon cold stimulation, contributing to the majority of beige adipocytes found in adult animals. Upon prolonged cold exposure, a small group of beige adipocytes can also emerge de novo. Focusing on the postnatal window, we identified ICAM1+/IL1R1+ committed preadipocytes as the precursors of postnatal beige adipocytes. This cell type appears to be bipotential, capable of giving rise to both beige and white adipocytes. Their thermogenic fate is primarily driven by a postnatal surge in circulating thyroid hormone, likely mediated through thyroid hormone receptor beta. We also revealed that macrophages can accelerate the lineage progression of these progenitor cells and increase the abundance of beige precursor cells. In adulthood, we examined the function and transcriptional profile of inactive beige adipocytes in both lean and obese animals. We found that inactive beige adipocytes persist within adipose tissue, even after long-term high-fat diet feeding. Although morphologically similar to white adipocytes and lacking thermogenic gene expression, inactive beige adipocytes exhibit distinct transcriptional profiles compared to classical white adipocytes. Functionally, we discovered that inactive beige adipocytes are linked to a lower occurrence of crown-like structures during obesity, a hallmark typically associated with chronic inflammation in white adipose tissue. This functional difference between inactive beige and white adipocytes is dependent on CCR2-mediated monocyte-recruitment, likely triggered by extracellular matrix remodeling driven by local adipocyte subtypes. Together, our findings offer important insights into the developmental and functional complexity of beige adipocytes, from their origins and molecular identity to their role in shaping the tissue microenvironment. This deeper understanding of beige adipocyte biology provides a foundation for developing new strategies to target this cell type in obesity and comorbid diseases
Mechanistic Insights into the Microtubule-Mediated Differential Modulations of Aurora B Substrate Structures for Accurate Chromosome Segregation
No full textFaithful chromosome segregation during eukaryotic cell division depends on the ability of sister kinetochores on duplicated chromatids to attach microtubules from opposite spindle poles. Failure in this process causes aneuploidy and drives cancer formation. To maintain genomic integrity, aberrant kinetochore-microtubule attachments must be destabilized, whereas properly attached microtubules must be stabilized. This process is regulated by Aurora B, the kinase subunit of the Chromosomal Passenger Complex (CPC). How Aurora B can distinguish between erroneous and correct attachments and selectively phosphorylate its key substrates in the right contexts remains poorly understood. The Ndc80 complex (Ndc80C) is a key substrate of Aurora B for regulating kinetochore-microtubule attachments. Ndc80C with Hypo-phosphorylated Ndc80 subunit (Hec1 in humans) binds microtubules with high affinity, promoting stable kinetochore-microtubule attachments; Phosphorylation at multiple sites within the Hec1 N-terminal tail weakens this interaction, promoting detachment for error correction. To investigate how phosphorylation of Ndc80C is controlled by microtubule binding, I developed a cryo-EM processing pipeline that resolves key phosphorylation sites of the Ndc80C in its microtubule-bound form. The resolved structure shows the disordered Hec1 tail domain with multiple Aurora B phosphorylation sites, engages in multivalent interactions to support the oligomerizing conformation of Ndc80C on microtubules. This geometry reveals a microtubule-mediated substrate masking mechanism, in which key phosphorylation sites become inaccessible to the kinase, thereby limiting phosphorylation. Combining with functional analyses, I propose that oligomerization of Ndc80 complex enhances its microtubule binding affinity and confers resistance to Aurora B-mediated phosphorylation. This substrate masking mechanism explains how stable kinetochore-microtubule attachments can be resistant to Aurora B triggered detachment. To determine whether substrate masking is a general feature of microtubule-bound Aurora B substrates, I examined mitotic centromere-associated kinesin (MCAK), a microtubule depolymerase whose activity is suppressed by Aurora B phosphorylation to stabilize spindle microtubules. Using the same cryo-EM pipeline, I resolved the structure of microtubule-bound MCAK and found that the key Aurora B phosphorylation site remains accessible when MCAK is bound to microtubules. This indicates that substrate masking is not a universal property of microtubule binding but is instead dependent on the oligomeric conformation specific to Ndc80C. Finally, I resolved the structure of the CPC on microtubules and found that it binds via the single α-helix domain of the INCENP subunit in a conserved manner across species. The CPC and MCAK partially share overlapping microtubule-binding sites, and cryo-EM-based co-decoration assays demonstrate that the CPC competitively displaces MCAK from the microtubule lattice. Given that microtubule binding of the CPC enhances Aurora B kinase activity, these suggest microtubules can promote Aurora B mediated phosphorylation of MCAK on microtubule to inhibit the depolymerization. In summary, the structural study described here demonstrates that microtubules can act as a signaling platform by differentially modulating the substrate accessibility of the Aurora B kinase to orchestrate chromosome dynamics during mitosis for faithful chromosome segregation
Biochemical and Structural Insights into VCP Unfoldase and WRN Helicase Complexes
No full textValosin-containing protein (VCP or p97 in mammals, Cdc48 in yeast) is an essential member of the AAA (ATPases associated with diverse cellular activities) protein superfamily. As its family name suggests, VCP function is implicated in a broad range of biological pathways including protein degradation, DNA repair, and organelle membrane remodeling. VCP acts as a mechanoenzyme by converting the energy from ATP hydrolysis into mechanical work to segregate its protein substrates from macromolecular complexes, membranes, or organelles and unfold them. This activity is regulated by interactions with cofactors, of which there are ~30 reported to engage VCP at distinct sites for the recruitment of substrates, localization to a subcellular compartment, and modulation of ATPase or unfoldase activity. VCP overexpression or mutation is linked to cancer and neurodegeneration, respectively, underscoring the critical role of VCP in maintenance of proper cell function. Decades of research have focused on the biochemical and structural principles of VCP function, especially pertaining to its role in the proteostasis network by unfolding polyubiquitylated substrates prior to their recycling or proteasomal degradation. For example, several groups have used structural biology approaches to determine structures of VCP in complex with cofactors and unfolded substrate. Through analysis of these assemblies, we have gained important insight into how substrate unfolding is initiated. However, structure determination is limited to a subset of cofactors that bind and recruit substrates and have a well-established biological role. The body of work contained in my thesis describes my efforts, in collaboration with others, to identify and characterize proteins that interact with VCP site-specifically with the goal of understanding how these interactions impact VCP\u27s role in diverse pathways. Chapter 1 serves as an introduction to proteostasis and the pathways involved in maintaining functional levels of proteins in cells. I also provide examples of how proteins in the proteostasis network have been targeted for the treatment of diseases. Additionally, I discuss the role of ubiquitylation, a post-translational modification with diverse functional implications, in the proteostasis pathway and include examples of small molecules that target enzymes within the ubiquitylation cascade. Finally, I introduce VCP\u27s structure, biochemical activities, regulation by cofactors, and roles in distinct cellular pathways. I describe studies that associate VCP overexpression or mutation with cancer and neurodegenerative diseases, respectively, and end Chapter 1 by summarizing reports that have identified candidate VCP binding partners using proteomics-based approaches. In Chapter 2, I describe my efforts to establish a method combining amber suppression, photo-crosslinking, and quantitative proteomics that identifies domain-specific VCP interactors. Interestingly, we identify profilin-1 (PFN1), an actin monomer-binding protein, as a top hit in one of our proteomics datasets. We examine the interaction between VCP and PFN1 using a biochemical pulldown assay and computational modeling. In collaboration with Eric Vitriol, we show that VCP can regulate actin polymerization in a PFN1-dependent manner. I also discuss progress toward characterization of a VCP-USP19 complex, which is another potential interaction discovered using our proteomics workflow. Chapter 3 contains a second project that developed from our chemical proteomics experiments, in which we observe VCPIP1, a deubiquitinase, bound to two distinct sites on VCP. Using cryo-electron microscopy, we determined structures of VCP-VCPIP1 complexes in the absence of added nucleotide or in the presence of an ATP analog and observe VCPIP1\u27s catalytic site below VCP\u27s central pore, poised to cleave ubiquitin from substrates following unfolding. We also show that VCP stimulates VCPIP1\u27s deubiquitinase activity in a biochemical assay. Together, these data suggest a model in which the activities of VCP and a deubiquitinase can be coupled to remodel substrates for recycling or degradation. In Chapter 4 I turn my focus to a project that I afforded significant time and energy, in which I examined the biochemical and structural properties of WRN helicase in complex with DNA substrates. I describe multiple efforts toward structure determination of WRN-DNA assemblies and additional studies where I examined the mechanism of action for a clinical-stage small molecule WRN inhibitor. While I have accumulated a large body of work focused on WRN, this project has not progressed to a publication. In the final chapter, I provide conclusions formed from the findings presented in this thesis and highlight potential directions for future studies. For example, I propose additional chemical proteomics experiments that could provide insight into how the VCP interactome may be altered in distinct cell states, such as disease or stress. I also suggest experiments toward structure determination of specific VCP complexes relevant to the findings presented herein. Finally, I discuss optimizations that may improve efforts toward structure determination of WRN-DNA complexes. In summary, this thesis captures the biochemical and structural properties of VCP unfoldase complexes in the context of proteostasis and cytoskeleton organization and highlights my progress toward the characterization of WRN-DNA-inhibitor interactions